One major obstacle for HIV vaccine development is the enormous genetic variability of viral strains between and within individuals. Recently, universal HIV-1 vaccine approaches have been proposed that exclusively target the highly conserved regions of the viral proteome, in which mutations are predicted to severely compromise virus viability. The underlining hypothesis is that a single mutation at a highly conserved amino acid site can be lethal or nearly lethal to the infecting virus and that the occurrences of compensation for these mutations are rare, e.g., multiple and/or fitness-reducing secondary mutations are required for compensation. However, the lethality of mutations at highly conserved amino acid sites has yet to be shown. In addition, we have found evidence indicating that the deleterious effect of mutations at highly conserved sites may be compensated for by mutations at variable sites. We define amino acid sites that are over 98% conserved in HIV-1 group M as highly conserved sites (HCS) and others as variable sites (VS). We propose a pilot exploratory study to test the underlining hypothesis of this vaccine approach. Using site-directed mutagenesis and in vitro growth competition assays, we will examine the lethality of naturally occurring Gag and Env mutations at 20 HCS (one mutation at each HCS) observed in an HIV-1 subtype B infected subject (PIC1362). Our objectives are: in PIC1362, 1) determine the lethality of the observed HCS mutations in the Gag and Env proteins, 2) determine the compensatory effect of the observed VS mutations;and 3) in two other HIV-1 infected subjects (PIC1113 and PIC1693), determine the lethality of Gag HCS mutations observed in PIC1362, to provide initial evidence of the generality of our findings in PIC1362. The results will provide new insights into the critical role of highly conserved amino acid sites in HIV-1 structure and function and help define the proteome constituents for inclusion in a conserved-elements HIV vaccine. PUBLIC HEALTH RELEVANCE: A universal HIV-1 vaccine design exclusively targeting the highly conserved regions of the viral proteome may present a novel way to tackle the enormous diversity in the HIV-1 viral population. This proposal tests the underline hypothesis of this approach towards HIV-1 vaccine development.